全人源抗肝癌噬菌体单链抗体筛选及鉴定

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1、全人源抗肝癌噬菌体单链抗体筛选及鉴定作者：黄建，肖艳，童永清，郭锋杰，周国华，李跃辉,胡锦跃，李官成【摘要】目的：构建人源抗肝癌噬菌体单链抗体库，从中筛选抗肝癌单链抗体(scFv),并对其特异性进行鉴定。方法：采用体外致敏法和EBV转化技术联合噬菌体展示技术构建噬菌体抗体库。对初级抗体库进行亲和富集及ELISA筛选，获得的阳性克隆进行免疫组化鉴定并测序。结果：scFv基因与载体连接后得到库容量为1.0X108的初级噬菌体抗体库。对抗体库进行3轮正负淘选和富集后，随机挑取2798个克隆进行ELISA,发现3个克隆

2、对HepG2呈强阳性反应，而与QSG7701等人正常细胞系呈弱阳性或不反应。对克隆SA3进行免疫细胞化学鉴定，结果与ELISA一致。免疫组织化学鉴定表明SA3与肝癌组织和肝组织阳性率的差别有统计学意义。结论：构建了库容量达1X108的全人源抗肝癌噬菌体抗体库。通过淘选富集、ELISA和免疫组织化学鉴定获得特异性较强的噬菌体克隆，为肝癌的临床诊断及导向治疗奠定了基础。【关键词】EE病毒转化噬菌体抗体库肝肿瘤单链抗体[Abstract]AIM：Toconstruetafullyhumanantihepatomasi

3、nglechainphageantibodylibrary,selecttheantihepatomascFvfromitandidentifyitscharacteristics.METHODS：AfullyhumanscFvdisplayingphagelibrarywasconstructedbyusingphageantibodylibrarytechniqueincombinationwithinvitroimmunizationandEBVtransformation.Thelibrarywassu

4、bjectedtothreeroundsofpositiveandnegativecellpanningandenrichmentandthenitwasselectedbyELISA.Thebindingspecificityofphageantibodieswithhepatomacarcinomacellswasconfirmedbyimmunohistochemistry.RESULTS：AfterscFvgenesbeingclonedintovectorFuse5andtransfonnedinto

5、E.coliMCI061viaelectroporation,aphageantibodylibrarycontaining1.0X108TU(transducedunit)wasobtainedthroughtetracyclineresistantscreening.Afterpanning,enrichmentandtestingbyELISA,3phageantibodyclonesreactingmorestronglytoHepG2thanQSG7701werepickedoutof2798clon

6、es.Oneclone,SA3,wasfurtheranalysedafterDNAsequencing・TheresuItsofimmunohistochemistrywithculturedcellsweresimilartothoseofELISA・SA3reactedspecificallytohepatomacellsinmosthumanhepatomatissuesectionsbutinfewhumannormallivertissuesections.Thedistinctionofposit

7、iveratesisofagreatstatisticalsignificance・CONCLUSION：AfullyhumanantihepatomascFvfusionphagelibraryhasbeenconstrueted.ELISAandimmunohistochemistryresuItsconformSA3specificallybindwithhepatomacarcinomacells.ThescFvfragmentagainsthepatomamaybefurtherdevelopedan

8、dappliedinclinicaldiagnosisandtherapyoflivercancer.[Keywords]EBVimmortalization；phageantibodylibrary；hepatoma；scFv肝癌是一种恶性程度极高的肿瘤，是我国和亚洲地区的常见病和多发病，病死率居恶性肿瘤的第3位[1]。肝癌90%以上为原发性，发病隐匿，发现时多数已处于中晚期,丧失了